The long term objective of this proposal is to advance the understanding of mechanisms of pulmonary oxygen toxicity and cellular mechanisms used to combat them by studying the relationship between antioxidants and heat shock proteins (HSP's) during hyperoxic stress. HSP's constitute a specific set of proteins induced by a large number of stresses. They are believed to aid in cell recovery from the stress. Their induction by hyperoxia has not been previously examined. Specific aims of this proposal include: 1) measuring the inducibility of HSP's by hyperoxia in vitro and in vivo; 2) assessing the degree of cross tolerance between heat stress and hyperoxic stress and how antioxidant levels may effect this; and 3) determining the relationship between antioxidant levels and the time course of induction of HSP's. Newborn and adult rats and newborn hamsters will be used for in vivo studies and fibroblasts isolated from their lungs used for in vitro studies since newborn rats are more resistant to hyperoxia than adult rats and newborn hamsters. In vitro studies are included because they constitute a more defined system and can be manipulated more easily which provides complimentary information. Animals will be exposed to a hyperoxic environment and induction of HSP's in the lungs determined by northern analysis using a 29-mer specific for the inducable form of HSP 70. This will be followed by a combination of in situ hybridization and immunocytochemistry to localize the cell type(s) in the lung producing HSP's. Induction of HSP's by hyperoxia in cultured lung fibroblasts from these same groups will be examined by northern analysis as above and by autoradiograms of proteins following radiolabeling and polyacrylamide gel electrophoresis. Stresses other than heat shock that induce HSP's have shown at least partial cross tolerance with heat shock and antioxidant levels have been demonstrated to effect thermal sensitivity in vitro. Cross tolerance between heat and hyperoxic stress and the effect of antioxidants will be tested in vitro by incubating cells with and without inhibitors of antioxidants. The time course of induction of HSP's may depend on the level of antioxidant protection. Antioxidants levels will be inhibited and enhanced in vitro and in vivo followed by hyperoxic exposure and analyses as described above to test this possibility. These studies will provide a framework for further investigations into mechanisms of pulmonary oxygen toxicity and provide a rational basis for the design of future therapy.